D1-like dopamine receptors have been implicated in CNS function and psychiatric disorders including the therapy of Parkinson's disease, schizophrenia, and drug abuse. Current therapeutic agonists that target D1 receptors are very limited and have potentially serious side effects. We hypothesize that developing a strategy that mimics the natural pulsatile activation of D1 dopamine receptors (activity- dependent) would offer a significant advantage for treating disorders associated with hypodopaminergic function of D1 receptors. To test this hypothesis it is necessary to develop positive allosteric modulators of D1 dopamine receptors that could be used to enhance the agonist-stimulated receptor signal. Identifying scaffold molecules to initiate this process was the primary goal for a recently completed high throughput screening (HTS) assay that was funded through the NIH Molecular Libraries and Imaging Roadmap Initiative. The specific aims described below will take five of the identified lead compounds as scaffolds for the synthesis and molecular characterization of putative allosteric modulators of D1 dopamine receptors. Specific Aim 1 will use the five previously identified lead probes to guide the synthesis of novel structural analogs. Specific Aim 2 will initiate the pharmacologic characterization of novel compounds in cells expressing D1 dopamine receptors. Each of the newly synthesized compounds will be tested for allosteric activity in a potentiator assay and a fold-shift analysis. Subsequent studies will also explore the potential signaling mechanisms of the test compounds. Specific Aim 3 will complete secondary assays to identify the potential sites of action and the mechanisms for the newly identified allosteric modulators of D1 dopamine receptors. These studies will include a series of radioreceptor binding studies, a receptor-specificity analysis, and mechanistic signaling studies. The studies in each of the Specific Aims will be iterative and the results from Specific Aims 2 and 3 will be used to drive the synthetic work proposed in Specific Aim 1. In summary, we propose to identify and characterize a novel class of chemical probes that can be used to modulate D1 dopamine receptor signaling. PUBLIC HEALTH RELEVANCE: Dopamine receptors are critical neuroreceptors that are involved in motor function, cognition, and addictive behaviors. The D1 dopamine receptor has been implicated as a target in the treatment of Parkinson's disease, schizophrenia, and drug abuse. Because current therapeutic agonists that target D1 dopamine receptors are very limited, we propose to identify a novel class of chemical probes that can be used to safely and effectively modulate D1 dopamine receptors to improve human health.